A critical step in the retroviral replication cycle is integration of a DNA copy of the viral genome into a chromosome of an infected cell. The objectives of this project are to understand the detailed molecular mechanism of this step in the retroviral replication cycle. In vitro assay systems developed in this laboratory have enabled us to previously determine many key features of the reaction mechanism. Our work continues to focus on the biochemical activities of HIV-1 integrase protein. The HIV-encoded integrase protein catalyzes both the 3' processing reaction that cleaves two nucleotides from the 3' ends of the viral DNA prior to integration, and also the subsequent DNA strand transfer step that inserts the viral DNA ends into a target DNA; integrase can also promote an apparent reversal of the DNA strand transfer reaction termed disintegration. We have constructed an extensive set of point mutations in the HIV integrase gene and purified the mutant proteins. Each protein has been assayed for 3' processing, DNA strand transfer, and "disintegration" activities. The results demonstrate that the central core region of integrase is both necessary and sufficient for the disintegration reaction and therefore contains the active site for polynucleotidyl transfer. However, additional functions supplied by the N-terminal and C-terminal regions of integrase are necessary for the 3' processing and DNA strand transfer activities. Mutant integrase proteins that have little or no activity when assayed alone complement to restore near wild type levels of activity when assayed as certain pairwise mixtures. This result demonstrates that integrase functions as a multimer in both the 3' processing and DNA strand transfer reactions. Inspection of the pairs of mutant proteins that are able to complement reveals that, within the active multimer, the N-terminal domain of one monomer functions together with the catalytic domain of another monomer.